Pre-analytical challenges from adsorptive losses associated with thiamine analysis

Thiamine (vitamin B1) is an essential vitamin serving in its diphosphate form as a cofactor for enzymes in the citric acid cycle and pentose-phosphate pathways. Its concentration reported in the pM and nM range in environmental and clinical analyses prompted our consideration of the components used in pre-analytical processing, including the selection of filters, filter apparatuses, and sample vials. The seemingly innocuous use of glass fiber filters, glass filter flasks, and glass vials, ubiquitous in laboratory analysis of clinical and environmental samples, led to marked thiamine losses. 19.3 nM thiamine was recovered from a 100 nM standard following storage in glass autosampler vials and only 1 nM of thiamine was obtained in the filtrate of a 100 nM thiamine stock passed through a borosilicate glass fiber filter. We further observed a significant shift towards phosphorylated derivatives of thiamine when an equimolar mixture of thiamine, thiamine monophosphate, and thiamine diphosphate was stored in glass (most notably non-silanized glass, where a reduction of 54% of the thiamine peak area was observed) versus polypropylene autosampler vials. The selective losses of thiamine could lead to errors in interpreting the distribution of phosphorylated species in samples. Further, some loss of phosphorylated thiamine derivatives selectively to amber glass vials was observed relative to other glass vials. Our results suggest the use of polymeric filters (including nylon and cellulose acetate) and storage container materials (including polycarbonate and polypropylene) for thiamine handling. Losses to cellulose nitrate and polyethersulfone filters were far less substantial than to glass fiber filters, but were still notable given the low concentrations expected in samples. Thiamine losses were negated when thiamine was stored diluted in trichloroacetic acid or as thiochrome formed in situ, both of which are common practices, but not ubiquitous, in thiamine sample preparation.

hour at 4˚C or 21˚C in a.) 8x40 mm 1 mL amber glass and polypropylene autosampler vials, 1.5 mL clear non-silanized and silanized glass autosampler vials, and b.) polypropylene and polystyrene 15-mL centrifuge tubes, polypropylene 1.5 mL centrifuge tubes, glass culture tubes (10x75 mm), and high-density polyethylene 125 mL bottles as compared to a 1 L stock solution stored at 21˚C in a glass media bottle.The results are after conversion of the thiamine remaining in solution to thiochrome using alkaline ferricyanide with fluorescence detection at λ ex = 360/40 nm, λ em = 450/50 nm.A vertical dashed line separates materials that may be directly compared based on their surface-to-volume ratios.*The inner surface area was calculated using the formula 2πrh for the cylinder walls in contact with the fluid, plus the formula 2πr 2 that of a hemisphere for the lower rounded tube bottom.The initial thiamine concentration was 100 nM, equating to 30, 50, and 75 pmol present in the 300, 500, and μL volumes, respectively.Recovery of thiamine in the solution following storage of 1 mL 100 nM thiamine, TMP, or TDP for 3 hours at 21˚C in HPLC grade water.A.) Amber glass, clear non-silanized, clear silanized glass, and polypropylene HPLC vials b.) polypropylene and polystyrene 15-mL centrifuge tubes, polypropylene 1.5 mL centrifuge tubes, glass culture tubes (10x75 mm), and high-density polyethylene 125 mL bottles were compared to the stock solution stored at 21˚C in a 50 mL polypropylene tube.The results are taken from the solutions after conversion to thiochrome using alkaline ferricyanide with detection in a microplate reader at λ ex = 360/40 nm, λ em = 450/50 nm.Filter Calculations: The filter calculations were done as follows: Thiamine standards were prepared (0-1000 nM) and a 50 μL volume was added to microwell plates, then converted to thiochrome using 100 μL alkaline ferricyanide for a total volume of 150 μL.The filters were pulled dry under vacuum and homogenized in 2 mL alkaline ferricyanide and 100 μL added to the wells, along with 50 μL water for a total volume of 150 μL.The volume and volume ratio of alkaline ferricyanide to aqueous solution was the same between samples and standards.The calibration curves and sample data were processed on the basis of pmol thiamine to account for the volume differences of sample to standard for the filter samples, then converted to concentrations.

Fig
Fig. S2.Impact of time on losses to silanized glass vials.The thiamine concentration recovered following storage of 1 mL 100 nM thiamine for 0 to 60 minutes in clear 1.5 mL silanized glass HPLC vials.The results are after conversion of the thiamine remaining in solution to thiochrome using alkaline ferricyanide with fluorescence detection at λ ex = 360/40 nm, λ em = 450/50 nm.

Fig. S3 .
Fig. S3.Levels of 300, 500, and 750 µL fluid in 10x75 mm and 12x75 mm borosilicate glass culture tubes.For illustration purposes, a dilute sulforhodamine B solution in HPLC grade water providing a magenta color was used in this picture.
Fig. S4.Thiamine concentration recovered from glass and plastic tubes after storage in TCA.Fluorescence of 100 nM thiamine in 7.5% (w/v) TCA after storage in 10x75 mm and 12x75 mm Type I borosilicate glass tubes, 5 mL polystyrene tubes, and 1.5 mL polypropylene tubes under static or vortexed conditions for 1 hour at 21°C.The tubes were vortexed moderately every 10 minutes for 10 seconds.The results are after conversion to thiochrome using alkaline ferricyanide with detection at λ ex = 360/40 nm, λ em = 450/50 nm.

Fig
Fig. S8.Glass and plastic filtration apparatuses Fig. S10.Autofluorescence and specific signals from filters A.) raw fluorescence intensities of 47 mm GF/F glass fiber, polyethersulfone (PES), and cellulose nitrate filters homogenized in 2 mL alkaline ferricyanide following passage of water in the absence of thiamine.Water only treated with alkaline ferricyanide is included as a no-filter control.A one-way ANOVA was used to compare the results for filter recovery to the no-filter control, with the p values listed in the figure.B.) Blank (0 nM) versus 100 nM thiamine through cellulose nitrate and PES filters.Apparent recovery of thiamine from the filtrate of 200 mL deionized water containing 0 or 100 nM added thiamine following passage through 0.2 µm 47 mm PES or cellulose nitrate membranes.The results are after conversion of the thiamine remaining in solution to thiochrome using alkaline ferricyanide with fluorescence detection at λ ex = 360/40 nm, λ em = 450/50 nm.Error bars represent one standard deviation of triplicate thiochrome determinations of the filtrate.

Fig. S11 .
Fig. S11.Effect of flow rate on thiamine concentration recovered from GF/F filters.A 1 L solution of regular tap water containing 100 pM thiamine was filtered at 5 to 20 min./Lthrough GF/F filters.The results are after conversion of the thiamine remaining in solution to thiochrome using alkaline ferricyanide with fluorescence detection at λ ex = 360/9 nm, λ em = 450/15 nm.

Table S1 .
Impact of Type I borosilicate glass tube dimensions on thiamine loss Static

Table S2 .
Time to pass 200 mL 100 nM thiamine in deionized tap water through filters when vacuum was set at 600 mm Hg